Epitope‐tagged knockin mice reveal ligand‐selective internalization that explains differential desensitization of alpha2‐adrenergic responses in native sympathetic neurons

Abstract

Using a gene targeting approach, we previously generated a knock‐in mouse line, in which N‐terminal hemagglutinin (HA) epitope‐tagged α2Aadrenergic receptor (AR) expression was driven by the endogenous mouse α2AAR locus. This line made it possible, for the first time, to evaluate α2AAR endocytosis and α2AAR‐mediated inhibition of Ca2+ currents in native neurons in response to clonidine and guanfacine, two drugs commonly used in treating attention deficit and hyperactivity disorder (ADHD) and eliciting analgesia through actions on the α2AAR. Exploiting this line, we found that the accelerated desensitization to clonidine compared to suppression of Ca2+ currents by guanfacine paralleled a more marked receptor phosphorylation and endocytosis of α2AAR than guanfacine. Interestingly, however, both guanfacine and clonidine share the same efficacy in coupling receptor occupancy to activation of G proteins, based on findings from receptor binding and α2AAR‐ activation of GTPγS studies. Our data provide strong evidence showing functional relevance of differential regulation of GPCR endocytosis by different ligands on receptor‐mediated signaling in native cells, and may represent an important determinant of therapeutic strategies, i.e. desensitizing or non‐desensitizing signals, depending on the pathophysiology being targeted.